Protein phosphorylation is important in many cellular processes including oncogenic transformation. Opal suppressor tRNAs may mediate specialized aspects of protein phosphorylation through read-through in response to termination codons and simultaneous insertion of phosphoserine. We are investigating the structure and function of opal suppressor genes by molecular cloning. A chicken opal suppressor gene has been isolated and sequenced. It is encoded in a single genomic copy and has an unusual internal 5 feet promoter region. In vitro transcription experiments indicate that it has an active but weak promoter. Gene copy number and promoter strength together account for the presence of a low intracellular level of this tRNA. The human and rabbit genomes contain DNA sequences that hybridize to the chicken gene, which is being used as a probe to isolate the corresponding human and rabbit genes from DNA libraries. Evolutionarily conserved regions integral to gene expression will be identified through gene comparison.